Glutathione (γ-L-glutamyl-L-cysteinyl-glycine), a substance that occurs widely in organisms, is known to act as a coenzyme, and to exhibit detoxicating action in the liver. Therefore, glutathione is widely used as a product, starting material or intermediate for pharmaceuticals, health foods, cosmetics and the like.
γ-glutamylcysteine (γ-L-glutamyl-L-cysteine), an intermediate for the biosynthesis of glutathione as stated above, is a thiol compound expected to have a flavor-improving effect similar to that of cysteine, and to serve as a therapeutic drug for lifestyle-related diseases and Alzheimer's disease.
Glutathione is known to be biosynthesized in Escherichia coli by conversion of L-glutamic acid and L-cysteine to γ-glutamylcysteine by γ-glutamylcysteine synthetase (GSHI), followed by the addition of glycine to the γ-glutamylcysteine by glutathione synthetase (GSHII); the gshA gene, which encodes GSHI, and the gshB gene, which encodes GSHII, have been isolated from E. coli (Non-patent Documents 1 and 2).
As methods for production of glutathione, enzymatic methods and fermentative methods are known. As enzymatic methods, methods are known wherein E. coli cells are used as an enzyme source in the presence of L-glutamic acid, L-cysteine, glycine and a surfactant (Patent Documents 1 and 2). As fermentative methods, methods are known wherein wild strains or mutant strains of Saccharomyces cerevisiae and Candida utilis, and recombinant strains of E. coli, S. cerevisiae and Lactococcus lactis transformed with the gshA gene and the gshB gene, are used (Non-patent Documents 3 and 4); in all the fermentative methods, glutathione is reportedly intracellularly accumulated.
Meanwhile, it is reported that the wild strain of E. coli extracellularly accumulates glutathione temporarily in the late logarithmic growth phase, although the amount is small (Non-patent Document 5).
It is known that a mutant strain of E. coli with a reduced or lost activity of γ-glutamyl transpeptidase exhibits an increased amount of glutathione accumulated in the medium, compared with the wild strain (Non-patent Documents 6 and 7), and it is also known that strains lacking the γ-glutamyl transpeptidase gene (ggt gene) and the yliAB gene, which encodes the glutathione uptake protein, exhibit further increased amounts of glutathione accumulated, compared with the strain lacking the ggt gene only (Non-patent Document 8).
It is also known that the protein encoded by the ybiK gene regulates glutathione uptake into cells (Non-patent Document 9), and that the ybiK gene has formed an operon with the yliABCD gene (Non-patent Document 8).
Although it is known that the protein encoded by the E. coli cydDC gene has an activity to deliver intracellular glutathione to periplasm (Non-patent Document 10), it is not known that the amount of glutathione accumulated in the medium increases when the protein's activity is enhanced.
The E. coli yceE gene, marA gene, marB gene, acrA gene, acrB gene, emrA gene, emrB gene, emrE gene, ydhC gene, ydeA gene, emrK gene, emrY gene, emrD gene, yajR gene, yegB gene, yidY gene, yieO gene and yjiO gene are estimated to be drug transporting genes, and the activity has been confirmed in some of these genes (Non-patent Document 19).
The ygeD gene is known as the lysophospholipid transporting gene (Non-patent Document 11). The bcr gene is known as the bicyclomycin resistance gene (Non-patent Document 12). The cmr gene is known as the chloramphenicol resistance gene (Non-patent Document 13). The marR gene is known as a multidrug resistance gene (Non-patent Document 14). The evgA gene is known as a drug efflux gene (Non-patent Document 15). The function of the ydeE gene is unknown.
As stated above, no genes are known to encode a protein having an activity to transport intracellular glutathione to the outside of cells.
As methods for production of γ-glutamylcysteine, methods of production using a mutant yeast strain in which the glutathione synthetase gene destroyed or weakened have been reported (Non-patent Documents 16 to 18); however, in all these fermentation methods, γ-glutamylcysteine is reportedly accumulated in cells.
patent document 1: JP-A-60-27396
patent document 2: JP-A-60-27397
non-patent document 1: Appl. Environ. Microbiol., 44, 1444 (1982)
non-patent document 2: Agric. Biol. Chem., 47, 1381 (1983)
non-patent document 3: Appl. Microbiol. Biotechnol., 66, 233 (2004)
non-patent document 4: Appl. Microbiol. Biotechnol., 24, 375 (1986)
non-patent document 5: J. Bacteriol., 168, 109 (1986)
non-patent document 6: Appl. Environ. Microbiol., 47, 653 (1984)
non-patent document 7: J. Bacteriol., 169, 3926 (1987)
non-patent document 8: J. Bacteriol., 187, 5861 (2005)
non-patent document 9: FEMS Microbiol. Letters, 209, 81 (2002)
non-patent document 10: J. Biol. Chem., 280, 32254 (2005)
non-patent document 11: J. Biol. Chem., 280, 12028 (2005)
non-patent document 12: Gene, 127, 117 (1993)
non-patent document 13: J. Bacteriol., 178, 3188 (1996)
non-patent document 14: J. Bacteriol., 175, 1484 (1993)
non-patent document 15: Microbiology, 149, 2819 (2003)
non-patent document 16: Agr. Biol. Chem., 54, 3145 (1990)
non-patent document 17: Molecular Biology of the Cell, 8, 1699 (1997)
non-patent document 18: Biochinica et Biophysica Acta, 1395, 315 (1998)
non-patent document 19: J. Bacteriol., 183, 5803 (2001)